Determination of the real electrochemical surface areas of screen printed electrodes

ABSTRACT

A method is provided for determining a real electrochemical surface area of a working electrode (WE) of a screen printed sensor. A concentration of a mediator incorporated in a WE paste is determined. The diffusion coefficient of the mediator is then ascertained. A final real electrochemical surface area of the WE is then made.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/141,159 filedDec. 29, 2008. This application is also related to commonly ownedPCT/IB05/02351 filed May 24, 2005. Both applications are fullyincorporated herein by reference.

BACKGROUND

1. Field of the Invention

This invention relates generally to methods for determining the actualsurface area of an electrode, and more particularly to methods for thedetermination of the real electrochemical surface area of a workingelectrode of a screen printed sensor.

2. Description of the Related Art

The actual surface area of an electrode where electron exchange takesplace is termed the real or active electrochemical surface area. It isdifferent from the geometrical surface area which is simply the sum ofall the physical areas that cover the surface of the electrode. Theratio of the electrochemical surface to the geometrical surface isrepresented by the roughness factor (ρ) as a coefficient. In general,the active area of an electrode exceeds the geometrical surface areacreating a higher number of chemically reactive sites on comparativelysmall electrodes. Most of the kinetic parameters of the electrodereaction as well as the electrical double layer properties need to bereferred to the unit area. Therefore, the determination of the realsurface area of the electrodes plays a crucial role in the calculationof various parameters characterising electrochemical processes andfacilitates quality control of mass-produced electrodes.

Various electrochemical methods to determine the real surface area ofconventional solid electrode (e.g. carbon electrodes and metalelectrodes) have been reported. Methods have been reported for thedetermination of real electrochemical surface area of liquid electrodes.By contrast, however, the determination of the real electrochemicalsurface area of screen-printed electrodes still remains underdeveloped,due to the inherently physical properties of screen-printed electrodes.For example, the surface of the screen-printed carbon electrodes is notas smooth as some conventional electrodes such as glassy carbonelectrodes and pyrolytic graphite electrodes. Thus, some electrochemicalmethods are inapplicable to the real surface area determination ofscreen-printed electrodes where the good surface condition is required.In addition, there are some other hindrances such as the complexity andnon-uniformity of the materials and production procedures forscreen-printed electrodes. To the best of our knowledge, no effectivemethod has previously been reported for the real electrochemical surfacearea determination of screen-printed electrodes.

Accordingly, there is a need for methods that determine the realelectrochemical surface area of screen-printed electrodes.

SUMMARY

An object of the present invention is to provide methods for determiningthe real electrochemical surface area of screen-printed electrodes.

Another object of the present invention is to provide methods fordetermining the real surface area of the electrodes in order todetermine various parameters characterising electrochemical processes.

Still another object of the present invention is to provide methods fordetermining the real surface area of electrodes to ascertain theconcentration range of the detected analytes.

A further object of the present invention is to provide methods fordetermining the real electrochemical surface area of screen-printedelectrodes using chronocoulometry.

Yet another object of the present invention is to provide methods fordetermining the real electrochemical surface area of screen-printedelectrodes by relying on the Anson equation which defines the chargetime dependence for linear diffusion control.

Still a further object of the present invention is to provide methodsfor determining the real electrochemical surface area of screen-printedelectrodes by calculating a diffusion coefficient and an unknownconcentration of the mediator incorporated in a working electrode paste.

These and other objects of the present invention are achieved in, amethod for determining a real electrochemical surface area of a workingelectrode (WE) of a screen printed sensor. A concentration of a mediatorincorporated in a WE paste is determined. The diffusion coefficient ofthe mediator is then ascertained. A final real electrochemical surfacearea of the WE is then made.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overall flowchart illustrating one embodiment of thepresent invention for determining the real area of the electrochemicalsurface area of a working electrode.

FIG. 2 is a flowchart illustrating one embodiment of the diffusioncoefficient used with the FIG. 1 embodiment.

FIG. 3 is a flow chart illustrating one embodiment of the TMPDconcentration used with the FIG. 1 embodiment.

FIG. 4 is a flow chart illustrating one embodiment of the realelectrochemical surface area of the working electrode of the presentinvention.

FIG. 5 illustrates a graph of signal to noise ratio.

FIGS. 6-8 are charts comparing signal to noise ratios for variousembodiments of the present invention.

FIGS. 9( a) and 9(b) illustrate embodiments of a testing deviceaccording an embodiment of the present invention.

FIG. 10 is a cross-sectional view of one embodiment of the presentinvention.

FIGS. 11( a) and 11(b) are schematics of electronic circuits accordingvarious embodiments of the present invention.

FIGS. 12( a) and 12(b) illustrate an embodiment of the present inventionusing as a switcher.

FIGS. 13( a)-13(c) illustrate another embodiment of the presentinvention using as a switcher.

FIG. 14 illustrates one embodiment of a disc for use with the presentinvention.

FIG. 15 is an exploded perspective view of one embodiment according tothe present invention.

FIG. 16 illustrates a ring according to an embodiment of the presentinvention.

FIG. 17 illustrates a cross-sectional view of one embodiment of thepresent invention.

FIG. 18 illustrates several embodiments according to the presentinvention.

DETAILED DESCRIPTION

Referring to FIGS. 1-4, in one of embodiment of the present invention, amethod is provided for determining the real surface area of ascreen-printed WE using chronocoulometry. In one embodiment, the WE isused in an analyte detecting system, such as one to determine aconcentration of glucose in the blood. Chronocoulometry relies on theAnson equation which defines the charge time dependence for lineardiffusion control. The Anson plot transforms the data into a linearrelationship whose slope is equal to 2nAFCD^(1/2)/π^(1/2)

The Anson plot is a plot of Q vs. t^(1/2) and transforms data into alinear relationship whose slope (a) is equal to:

2nAFCD ^(1/2) /π ^(1/2)

-   -   where:    -   Q is the charge (coulombs);    -   n is the number of electrons transferred;    -   A is the real electrochemical surface area of the WE (cm²);    -   F is Faraday's constant (96,485 coulombs/mole);    -   C is the concentration of the mediator (moll/cm³); and D is a        diffusion coefficient of the mediator (cm²/sec).

By calculating the diffusion coefficient and the unknown concentrationof the mediator incorporated in the WE paste, and by rearranging theabove equation, it is possible to determine the real electrochemicalsurface area of the WE of a screen printed sensor.

The method of the present invention facilitates determination of thereal surface area of screen-printed WE's and provides betterfunctionality, as well as providing an improvement of a sensor.

In one embodiment, a method is provided for determining a realelectrochemical surface area of a WE of a screen printed sensor. The WEpaste can be a complex matrix structure containing both conductingelements and assay reagents.

A determination or calculation is made of a concentration of a mediatorincorporated in a WE paste. This determination can include runningcyclic voltammetry, and applying a standard addition method. In oneembodiment, different solutions of mediator with differentconcentrations in PBS are prepared, and cyclic voltammograms are thenrun to observe an (Eox) of the WE after applying increasedconcentrations of mediator on the WE. A specific electrochemicalresponse corresponds to an added concentration of the mediator as wellas of an unknown concentration of the mediator present in the WE paste.Oxidation peak currents observed in the cyclic voltammograms (“CVs”) canbe plotted against a concentration of the added mediator. Aftercalculating a slope and an intercept of the oxidation peak currents, theunknown concentration of the mediator is calculated by dividing theintercept with the slope.

In one embodiment, the concentration of the mediator incorporated in theWE paste is unknown. A calculation or determination is made of thediffusion coefficient of the mediator. A determination or calculation isthen made of the final real electrochemical surface area of the WE. As anon-limiting example, the screen printed sensor can be a three electrodesystem that includes, two printed carbon electrodes acting as a WE andcounter electrode, and a printed Ag/AgCl reference electrode.

In one embodiment, the calculation of the diffusion coefficient of themediator includes first running cyclic voltammetry under three differentscan rates to create electrode cyclic voltammograms, and then plottingoxidation peak currents against a square root of scan rates. In oneembodiment, PBS is run on the electrode cyclic voltammograms under threedifferent scan rates to obtain three different oxidation peak currentsthat correspond to the different scan rates. In one embodiment, theslope of the plot of the three oxidation peak currents (Eox) plottedagainst the square root of the scan rates is equal to a diffusioncoefficient of the mediator.

Chronocoulometry can be used to determine the real electrochemicalsurface area of the WE. Chronocoulometry is a measurement of charge as afunction of time, wherein an analysis of chronocoulometric data is basedon an Anson equation that defines a charge-time dependence for lineardiffusion control:

Q=2nFACD ^(1/2)π^(−1/2) t ^(−1/2)

-   -   where:    -   Q is the charge (coulombs);    -   n is the number of electrons transferred;    -   A is the real electrochemical surface area of the WE (cm²);    -   F is Faraday's constant (96,485 coulombs/mole);    -   C is the concentration of the mediator;    -   D is a diffusion coefficient of the mediator (cm²/sec); and    -   t is time (sec).

EXAMPLE 1

In order to use the above equation is necessary to know theconcentration of the mediator in the WE paste as well as the diffusioncoefficient of the mediator. To calculate the diffusion coefficient ofthe mediator, cyclic voltammetry was employed. By applying PBS on theelectrode cyclic voltammograms were run in three different scan rates(10 mV/sec, 25 mV/sec and 50 mV/sec). Three oxidation peak currents(Eox) were then plotted against the square root of the scan rates. Theobtained slope of the above plot is equal to the diffusion coefficientof the mediator.

In order to calculate the unknown concentration of the mediator thestandard addition method was used. Five different solutions of TMPD wereused with different concentrations (100 μM, 250 μM, 500 μM, 750 μM and1000 μM) in PBS and cyclic voltammograms were run in order to observethe (Eox) of the WE after applying increased concentrations of mediatoron the WE. The specific electrochemical response corresponded to theadded concentration of the mediator as well as of the unknownconcentration of the mediator present in the WE paste. In order toobserve the electrochemical response of the sensor, which correspondsonly to the concentration of the mediator present in the WE paste,cyclic voltammograms were run with plain PBS. The oxidation peakcurrents observed in the CVs were then plotted against the concentrationof the added mediator. After calculating the slope and the intercept ofthe above plot the unknown concentration of the mediator was determinedby dividing the intercept with the slope.

The final values of the diffusion coefficient and TMPD concentrationused in the Anson equation were the average value of three replicates.

In one embodiment, the present invention provides optimized signal tobackground noise designs. Current ratio of signal to backgroundoptimization can use one of the following to obtain improved results: 1)use of a hydrogel with zwitterionic compounds or 2) changes inhydrophilic layer dimensions to obtain improved signaling. Currentlyavailable test strips have a maximum signal to noise ratio of about 20.Experimental data for analyte detecting devices with and withouthydrophilic layer and with/without zwitterionic compound illustratesthat the present invention can achieve significantly improved signal tonoise ratio. In other embodiments, the mediator content, geometricaldesign of the electrode and the sample chamber also influence thesignal.

Some embodiments can achieve signal to noise ratio greater than 20. Someembodiments can achieve signal to noise ratio greater than 25. Someembodiments can achieve signal to noise ratio greater than 30. Someembodiments can achieve signal to noise ratio greater than 35. In oneembodiment, because the reduced form of the mediator is embedded intothe reaction zone of one embodiment of the present analyte detectingdevice, a current can be observed after applying the voltage. Thisgenerates the oxidized form of the mediator, which is able to react withthe reduced form of the enzyme glucose-oxidase. For this particularexample, in the absence of glucose (using phosphate buffered saline only(pH 7.4)) a (background) current, will be detected.

As a nonlimiting example, adding 500 mM glucose in buffer and applying avoltage, the saturation current can be determined. The excess glucose isso that the device is not limited by lack of glucose when making thesaturation current measurement. The original background current can becaused by the oxidation of mediator by the electrode. In one embodimentof the present invention, the mediator exists in the reduced form in thecarbon paste, and is active in the oxidized form during the measurement.

There is typically always a little reduced mediator present, which givesthe background signal (which is termed the “glucose independent”current). Approaches to reduce this background current in the art are totry and oxidize the mediator before the measurement. Most areinefficient at achieving this and so there is a lot of reduced mediatoravailable leading to a larger background current and hence lower Q (inthe range of 20).

Chronoamperometic methods look at last section of the graph so that alow amount of reduced mediator is observed, as most of the mediator hasdisappeared from the electrode paste and does not contribute tobackground. coulometric methods look at charge generated during theentire reaction. At the start, there is a lot of background signal, andat the end there is little, but the result is the integration over thewhole time, resulting in total charge and hence the sum of backgroundsignal as a result. It is therefore inherently difficult with thecoulometric methods to get a high Q. The ratio of y previously,thickness of the hydrogel can also improve signal to noise ratio. Itshould be understood that chamber dimension and layer thickness mightinfluence the signal. Several micrometers in change to they hydrogellayer can be sufficient to alter the ratio. In one embodiment, thethickness of the layer is about 4 micrometers. Increasing the thicknessalso increases the ratio. Unfortunately, increased thickness of thelayer can also slow the diffusion rate of the analyte.

The slope is the diffusion dependent current, and if a thick hydrophilicmember is used, the diffusion rate of glucose to electrodes isdecreased. Number of glucose molecules per second is slower. In someembodiments, the thickness will not exceed 30 micrometers. In otherembodiments, the thickness will not exceed 50 micrometers.

Referring now to FIG. 5, the importance of a high Q value is reflectedin the ability to measure glucose at the higher concentrations. FIG. 5illustrates the results of glucose concentration versus current for twodifferent batches of test strips, which have components known togenerate different values of Q. As seen, the test strip batch having aratio of 23 has only a measuring range up to 20 mM glucose in wholeblood, whereas a batch having a ratio of 37 illustrates a measuringrange up to about 40 mM glucose in whole blood. Both batches have samebackground. (In one embodiment of the present invention, the backgroundcurrent is about 1000 nA).

The graph of FIG. 5 confirms the same precision in the low range ofglucose, but higher precision in the high range as well as a broaderrange of glucose concentration measurement at the high glucose levels.At low glucose concentration (2.5 mM) the ratio between the slope andthe background is important and the precision is important. Q can beincreased by applying (e.g. screen-printing) a hydrophilic membrane overthe working electrode.

FIG. 6 illustrates that application of a hydrophilic membrane to theanalyte detecting device construction increases Q. The membraneeffectively functions by enhancing the concentration of the mediator atthe surface of the working electrode. The net result is that the actualconcentration of the mediator in the reaction zone is higher incomparison to the batch having no hydrophilic membrane.

Referring now to FIG. 7, Q can be increased even further by optimizingthe formulation of the paste for the hydrophilic such as using of adetergent as part of the hydrophilic membrane. The magnitude of theincrease of course depends on the type of detergent used. The Q valuefor trips containing Triton or CHAPS are compared in FIG. 7. Using thezwitter-ionic detergent CHAPS in the hydrophilic membrane Q wasincreased by a factor of 1.5 in comparison to the non-ionic detergentTriton X-IOO. Referring now to FIG. 8, further modifications of analytedetecting devices according to the present invention lead to ratioshaving values up to 148, though the detergent tends to be ratherunstable. Different types of analyte detecting device constructions havegiven rise to different values of Q in FIG. 8.

Batch 1 is an analyte detecting device without membrane; similar to whatis available in the art the resultant Q is 23. Batch 2 is an analytedetecting device constructed with the proprietary hydrophilic membrane,increasing to Q to 37. Batch 3 and 4 contain the hydrophilic membraneand different zwitterionic detergent compounds, resulting in almostdouble of the value for Q. Batch 5 is the highest operating Q value ofabout 150, but the detergent has proven to be rather unstable. In oneembodiment, Q of the present invention is in the range of 60-80. 2)

In another embodiment, the present invention provides a method formanufacturing an analyte detecting device using screen printing aplurality of layers, such as but not limited to seven layers whereinadhesive is counted as one layer. Currently available glucose teststrips have sample chambers created by laminating step.

In one embodiment, the analyte detecting structures of the presentinvention can be formed by laying the following layers. 1. Conductivelayer 2. Insulating layer 3. Reference and counter electrode 4. WE 5.hydrophilic membrane 6, spacer layer 7 and adhesive layer. FIG. 9( a)illustrates dimension of the sample capturing structure without mesh(GS-SC 1, in its original form). The structure, in some otherembodiments, can includes a mesh. By way of example and limitation, achannel 40 can have a width of 0.5 mm. The opening over each electrodeis also about 0.5 mm. The contact pads can have a size of 1.2 mm. Anopening can have a diameter of 1.0 mm. As seen in FIG. 9( b), oneembodiment of the present invention comprises of six layers which can bemanufactured by seven printing steps. In this method, five stepsconstruct the electrode elements 30 while 2 steps account for the twolayers 32 and 34 that comprise the microfilling features.

In one embodiment of the present invention, channels are printed thatare used for hydrophilic filling. 30 μm2 printing is challenging butbeing done. In one embodiment, a 30 μm2×50 μm thick electrode would useInl volume. In some embodiments of the present invention a sample volumeof only 0.6 μL is required. In one embodiment, the noise floor issuesmean that the lowest amperage for dilution can 10 nA. By way ofillustration, and without limitation, the dimensions are 0.4 μL, 5mm2×80 μm area×thickness. In one embodiment, 0.2 μL 4mm2×50 μm=200 nAsignal. In one embodiment, lmm2×100 μm=0.1 μL is challenging but doablefor a 10 nA signal, the present invention could go 1/20 of the 0.2 μL.In one embodiment, the present invention could theoretically do 0.1mm2×0.05 μL or 0.005 μL 0.5 nL.

Referring now to FIG. 10, an embodiment is illustrated connectinghydrophilic layer to the conductive layer. The “mushroom” shapedelectrode can connect the conductive layer to the hydrophilic membranewith increased surface area using a drop through geometry. Themushroom-shaped cap of the electrode is configured to increase thesurface area in contact with the layer above.

FIG. 10 illustrates a cross-section of the analyte detecting members. Inthis embodiment, a substrate 100 is provided. On top of this substrate,a carbon paste is provided to form conducting layers 102 for ascreen-printed three-electrode system. A spacer layer 104 can also beprovided. The reference and the counter electrodes 142 and 143 can bemade of a formulation of Ag/AgCl. The analyte detecting member can bebased on chrono-amperometry measurement technique using glucose oxidase(Gox) enzyme and N,N,N′, N′-Tetramethyl-p-phenylenediamine (TMPD), aselectron transfer mediator. Although not limited to the following, theworking electrode 140 can optionally comprise of carbon paste blendedwith Gox, the mediator, a buffer and a thinner.

A hydrophilic layer or membrane 108 is provided on top of theelectrodes. In some embodiments, only the working electrode 140 has thehydrophilic layer 108. It should be understood that the hydrogel can beformed in a variety of shapes including but not limited to rectangular,square, polygonal, circular, triangular, any single or multiplecombination of shapes, or the like. Embodiments of the present inventioncan use a three-electrode system. For testing devices with “bad”mediators, there is normally a dummy electrode, which is used to reducebackground current. The reference electrode of the present invention isa more advanced design as it conveys the potential. It is independent ofsample variability, so it achieves a constant potential between theworking electrode and the counter electrode.

Referring now to FIG. 11( a), another embodiment of the presentinvention provides a meter relay 200 for sensing sample arrival andmonitoring flow of sample in the testing device. In one embodiment ofthe present invention, FIG. 11( a) illustrates a system with electronicsthat do not handle switching and coupled to a testing device 150.

FIG. 11( b) illustrates a system with electronics that two of theelectrodes can used for an auto-trigger function while all threeelectrodes can be used for the measurement. FIG. 11( b) illustrates ananalyte detecting device 150 coupled to a switch 200. In thisembodiment, the switch is located in the analog portion of the meterelectronics. Other embodiments can locate the switch in a digital partof the circuit. A switch, relay or other type of general switch devicecan be used to switch monitoring of various electrodes. The switch 200allows for monitoring of the first and second electrodes which contactthe fluid sample. The switch 200 when moved into a second configurationallows for monitoring of the second and third electrodes in the testingdevice that contact the fluid sample. The signal is transferred to thedigital portion 203 of the circuit which includes a microcontroller 205for processing information from the various sets of electrodes.

As seen in the embodiment of FIG. 12( a), in the start condition, thecounter relay 200 is open as indicated by arrow 208. A referenceelectrode lead 210, counter electrode lead 212, and working electrode214 lead is illustrated. As seen in FIG. 8, if the blood sample wets thereference electrode as indicated by arrow 216, the measurement starts inthe two electrode-system modes until the current reaches the thresholdlevel. Then in FIG. 12( b), the counter relays 200 closes as indicatedby arrow 220 and the amperometric measurement for the detection of theglucose occurs in the three electrode-system mode. Threshold level(value for auto trigger) is adjustable from 50 to 2,000 Na. Thus, a twoelectrode-system used for auto-trigger function, three electrode-systemused for the measurement. In summary, blood sample wets the referenceelectrode, the counter relays is switched, and the amperometricmeasurement starts.

In one embodiment, instead of measurement using the first twoelectrodes, the electrodes can also be used to monitor fluid flow in thecapillary. As a non-limiting example, when blood covers the firstelectrode and contacts the second electrode, the signal begins to flow.The relay 200 then switches to monitor the time it takes for blood(after it contacts the second electrode, typically the workingelectrode) until the blood reaches the third electrode. The time ittakes for the blood to flow from working electrode to the thirdelectrode is monitored to know the flow velocity in the capillary. Ifthe time it takes for blood to flow from the second to third electrodeexceeds a threshold, then the measurement can be discarded. As anon-limiting example, the threshold can be one minute, two minutes, ormore.

Referring now to FIGS. 13( a)-13(c), another embodiment of the presentinvention will now be described. As indicated by arrow 230, in thestarting condition, the counter array 200 is closed. If the blood sampleB wets the working electrode as indicated by arrow 232 as seen in FIG.13( b), a current is detected, a non-regulated potential. The relays areswitched to the open modus as indicated by arrow 234. When a bloodsample wets the working electrode, the counter relay is switched to theopen modus. If the blood sample wets the reference electrode, themeasurement starts in the two electrode-system mode until the currentreaches the threshold level. The counter relay then closes, as indicatedby arrow 236, and the amperometric measurement for the detection of theglucose occurs in the three electrode-system mode.

This feature provides the opportunity to measure the flow rate of theblood. This feature also permits the system to reload with more blood.However, if the time difference (current peak, if working electrode hasbeen wet, and current peak, if reference electrode has been wet) is toolong, the measurement is omitted (time control for reloading). As anon-limiting example, the threshold level, value for auto trigger, canbe adjustable from 50 to 2,000 nA. The threshold level is a compromisebetween possible non-fluid caused currents and the slope. Although bloodis the example used herein for illustrative purposes, it should beunderstood that other body fluids can be used, depending on the analytebeing measured. When blood samples wet the reference electrode, thecounter relay is switched to the closed modus and the amperometricmeasurement begins.

In certain embodiments, the present invention not only detects that thecapillary is filled when blood reaches the third electrode, but it alsomonitors the movement of blood to and through the electrochemical cellor testing device. Detecting movement into and through the channel canhave its benefits. In one embodiment, the present invention monitors themovement of the sample. This can be achieved with detecting thepotential between two electrodes. There is a current flow when twoelectrodes are contacted by blood and a signal is generated. Thepotential is then between the two different electrodes, which allowscurrent flow and a signal. The second signal illustrates that thecapillary is filled. As discussed above, the present invention can use aswitcher. A general switcher can be used to monitor two differentlocations. In this embodiment monitoring means determining the entry ofthe blood, when the capillary is filled, and thus the velocity of fluidflow. This is based on the time.

In one embodiment, the electrochemical cell starts when touch workingelectrode and when capillary is filled. In one embodiment, the partialcovering of the working electrode and filling the whole channel areachieved. The allows the monitoring of blood through the sample device.A maximum time that is allowed to fill the whole channel can bedetermined.

FIG. 14 illustrates one embodiment of a cartridge 300 that can beremovably inserted into an apparatus for driving penetrating members topierce skin or tissue. The cartridge 300 has a plurality of penetratingmembers 302 that can be individually or otherwise selectively actuatedso that the penetrating members 302 can extend outward from thecartridge, as indicated by arrow 304, to penetrate tissue. In thepresent embodiment, the cartridge 300 can be based on a flat disc with anumber of penetrating members such as, but in no way limited to, (25,50, 75, 100, . . . ) arranged radially on the disc or cartridge 800. Itshould be understood that although the cartridge 300 is illustrated as adisc or a disc-shaped housing, other shapes or configurations of thecartridge can also work without departing from the spirit of the presentinvention of placing a plurality of penetrating members to be engaged,singly or in some combination, by a penetrating member driver. Eachpenetrating member 302 can be contained in a cavity 306 in the cartridge300 with the penetrating member's sharpened end facing radially outwardand can be in the same plane as that of the cartridge. The cavity 306can be molded, pressed, forged, or otherwise formed in the cartridge.Although not limited in this manner, the ends of the cavities 306 can bedivided into individual fingers, such as one for each cavity, on theouter periphery of the disc. The particular shape of each cavity 306 canbe designed to suit the size or shape of the penetrating member thereinor the amount of space desired for placement of the analyte detectingmembers 808.

As a non-limiting example, the cavity 306 can have a V-shapedcross-section, a U-shaped cross-section, C-shaped cross- section, amulti-level cross section or the other cross-sections. The opening 810through which a penetrating member 302 exits to penetrate tissue canhave a variety of shapes including but not limited to, a circularopening, a square or rectangular opening, a U-shaped opening, a narrowopening that only allows the penetrating member to pass, an opening withmore clearance on the sides, a slit or the other shapes. In thisembodiment, after actuation, the penetrating member 302 is returned intothe cartridge and can be held within the cartridge 300 in a manner sothat it is not able to be used again.

By way of example and not limitation, a used penetrating member can bereturned into the cartridge and held by the launcher in position untilthe next lancing event. At the time of the next lancing, the launchercan disengage the used penetrating member with the cartridge 300 turnedor indexed to the next clean penetrating member such that the cavityholding the used penetrating member is position so that it is notaccessible to the user, e.g., turned away from a penetrating member exitopening.

In some embodiments, the tip of a used penetrating member can be driveninto a protective stop that hold the penetrating member in place afteruse. The cartridge 300 is replaceable with a new cartridge 300 once allthe penetrating members have been used or at such other time orcondition as deemed desirable by the user. I S Referring still to theembodiment in FIG. 14, the cartridge 300 can provide sterileenvironments for penetrating members via seals, foils, covers,polymeric, or similar materials used to seal the cavities and provideenclosed areas for the penetrating members to rest in. In the presentembodiment, a foil or seal layer 320 is applied to one surface of thecartridge 300. The seal layer 320 can be made of a variety of materialssuch as a metallic foil or other seal materials and can be of a tensilestrength and other quality that can provide a sealed, sterileenvironment until the seal layer 320 is penetrate by a suitable orpenetrating device providing a preselected or selected amount of forceto open the sealed, sterile environment.

In one embodiment, each cavity 306 can be individually sealed with alayer 320 in a manner such that the opening of one cavity does notinterfere with the sterility in an adjacent or other cavity in thecartridge 800. As seen in the embodiment of FIG. 14, the seal layer 320can be a planar material that is adhered to a top surface of thecartridge 800. Depending on the orientation of the cartridge 300 in thepenetrating member driver apparatus, the seal layer 320 can be on thetop surface, side surface, bottom surface, or other positioned surface.For ease of illustration and discussion of the embodiment of FIG. 14,the layer 320 is placed on a top surface of the cartridge 800. Thecavities 306 holding the penetrating members 302 can be sealed on by aseal layer 320 and thus create the sterile environments for thepenetrating members. The seal layer 320 can seal a plurality of cavities306 or only a select number of cavities as desired.

In one embodiment, illustrated in FIG. 14, the cartridge 300 canoptionally include a plurality of analyte detecting members 308 on asubstrate 822 which can be attached to a bottom surface of the cartridge300. The substrate can be made of a material such as, but not limitedto, a polymer, a foil, or other material suitable for attaching to acartridge and holding the analyte detecting members 308.

The substrate 322 can hold a plurality of analyte detecting members,including but not limited to about, 10-50, 50-100, or other combinationsof analyte detecting members. This facilitates the assembly andintegration of analyte detecting members 308 with cartridge 300. Theseanalyte detecting members 308 provide an integrated body fluid samplingsystem where the penetrating members 302 create a wound tract in atarget tissue that spontaneously expresses body fluid that flows intothe cartridge for analyte detection by at least one of the analytedetecting members 308.

The substrate 322 can contain any number of analyte detecting members308 suitable for detecting analytes in cartridge having a plurality ofcavities 306. In one embodiment, many analyte detecting members 308 areprinted onto a single substrate 322 which is then adhered to thecartridge to facilitate manufacturing and simplify assembly. The analytedetecting members 308 can be electrochemical in nature. The analytedetecting members 308 can further contain enzymes, dyes, or otherdetectors which react when exposed to the desired analyte.

Additionally, the analyte detecting members 308 can have clear opticalwindows that allow light to pass into the body fluid for analyteanalysis. The number, location, and type of analyte detecting member 308can be varied as desired, based in part on the design of the cartridge,number of analytes to be measured, the need for analyte detecting membercalibration, and the sensitivity of the analyte detecting members. Ifthe cartridge 300 uses an analyte detecting member arrangement where theanalyte detecting members are on a substrate attached to the bottom ofthe cartridge, through holes, as illustrated in FIG. 11, wickingelements, capillary tube or other devices can be provided on thecartridge 300 to allow body fluid to flow from the cartridge to theanalyte detecting members 308 for analysis.

In some embodiments, the analyte detecting members 308 can be printed,formed, or otherwise located directly in the cavities housing thepenetrating members 302 or areas on the cartridge surface that receiveblood after lancing. The use of the seal layer 320 and substrate oranalyte detecting member layer 822 can facilitate the manufacture ofthese cartridges 10. For example, a single seal layer 320 can beadhered, attached, or otherwise coupled to the cartridge 300 asindicated by arrows 324 to seal many of the cavities 306 at one time. Asheet 322 of analyte detecting members can also be adhered, attached, orotherwise coupled to the cartridge 300 as indicated by arrows 325 toprovide many analyte detecting members on the cartridge at one time.

As a non-limiting example, during manufacturing of one embodiment of thepresent invention, the cartridge 300 can be loaded with penetratingmembers 302, sealed with layer 320 and a temporary layer (notillustrated) on the bottom where substrate 322 would later go, toprovide a sealed environment for the penetrating members. This assemblywith the temporary bottom layer is then taken to be sterilized. Aftersterilization, the assembly is taken to a clean room, or it can alreadybe in a clear room or equivalent environment, where the temporary bottomlayer is removed and the substrate 322 with analyte detecting members iscoupled to the cartridge as illustrated in FIG. 14. This process allowsfor the sterile assembly of the cartridge with the penetrating members302 using processes and/or temperatures that can degrade the accuracy orfunctionality of the analyte detecting members on substrate 322.

As a nonlimiting example, the entire cartridge 300 can then be placed ina further sealed container such as a pouch, bag, plastic moldedcontainer, and the like, to facilitate contact, improve ruggedness,and/or allow for easier handling. In some embodiments, more than oneseal layer 320 can be used to seal the cavities 306. As examples of someembodiments, multiple layers can be placed over each cavity 306, half orsome selected portion of the cavities can be sealed with one layer withthe other half or selected portion of the cavities sealed with anothersheet or layer, different shaped cavities can use different seal layer,or the like. The seal layer 320 can have different physical properties.In one embodiment, the seal layer covering the penetrating members 302near the end of the cartridge can have a different color to indicate tothe user that the number of penetrating members is running low.

As illustrated in FIG. 15, device 400 is illustrated in an explodedview. In this embodiment, device 400 includes a hydrophilic cover 402,spacer 404, hydrophilic member 406, working electrode 408, counter andreference electrodes 410, insulating layer 412, conductive lines 414,and a substrate 416. The analyte detecting members can be printed insheets. From the sheets the analyte detecting members are placed at highdensity in concentric rings or other configurations, including but notlimited to lines, linear arrays, circular arrays, square arrays,polygonal arrays, triangular arrays, and the like, on the disk.

Referring now to the FIG. 16 embodiment, the analyte detecting members420 are positioned in the arrays on sheets at high density. In oneembodiment, high density manufactured analyte detecting members are diecut and placed on the ring 450. The density of the analyte detectingmembers is increased by adhering the analyte detecting members inconsecutive rings that diminish in size as the diameter decreases, goingfrom the outside to the inside of the ring. This can decreasesmanufacturing cost 12 fold in comparison to GSD. As a non-limitingexample, in this embodiment the printed areas can be 150 sq mm comparedto 1875 sq mm for GSD.

Desiccant can also be printed in a paste that forms a microfluidicchannel. This reduces additional packaging. As a non-limiting example,the desiccant can be a desiccant-coated Al film

In another embodiment, illustrated in FIG. 17, an analyte detectingmember disc 580 can be mounted to position analyte detecting members 590on an outside rim of the cartridge 600 housing. At least one penetratingmember 602 is provided. In some embodiments, a hydrophilic cover member604 can be included. In the FIG. 17 embodiment, analyte detecting can beaccomplished using a film mesh 610 at a front end to draw blood to theanalyte detecting members 590. The film mesh 610 can be advanced afteruse to provide a fresh piece for every test. In the FIG. 17 embodiment,the cartridge 600 and analyte detecting member disk 580 are the same,contamination is reduced and/or eliminated, analyte detectingmember-analyte detecting member-isolation is achieved due to thehydrophilic cover film and analyte detecting member and penetratingmember isolation is achieved.

In one embodiment, contact to the meter is easily achieved through pinsto establish electrical contact between conductive lines coupled tomembers 590 and the meter (not illustrated). A vent can be created at abeginning of measurement by using a needle to pierce a hole through thefilm at the end of the capillary. A penetrating member exit point can bethrough a center of the film mesh 610. In one embodiment, a new indexingmechanism is provided to move the disc in addition to the cartridge 600.This provides dull rotation and indexing and allows for an increase inthe density of the analyte detecting members. As a non-limiting example,one analyte detecting disc can be used for every two penetrating membercartridges. Additionally, precise film transportation can be used.

In another embodiment, illustrated in FIG. 18, the height of the channelis decreased to decrease the volume of blood or fluid sample. As anon-limiting example, the height of the channel is decreased and thevolume of fluid sample was dropped to 0.2 μL from a starting samplevolume of 0.6 μL. As non-limiting examples, (i) the height of thechannel can be about 50 μm and (ii) the length of the channel is reducedand the height increased to 80 μm which minimizes problems of highHaematocrit or viscosity. As a non-limiting example, the sample volumeis 0.4 μL, width of the channel is 500 μm by masking, the channel has alength of 1000 μm, 4 layers above the electrode are aligned, the workingelectrode is 2000 μm and has a 500 μm gap. As a non-limiting example,the size of the sample capturing structure in its original form is 6.15mm×5 mm (without mesh, see FIG. 5A, as well as 7.75 mm×5mm (with mesh).

FIG. 18 illustrates an overview of the three different variants ofsample capturing structures that has an enlarged structure.

Referring to FIG. 17, in various embodiments dimensions of differentlayers and structures are as follows: (i) GS-SC 1 does not have a mesh620 and both apertures have a diameter of 1 mm, (ii) GS-SC 2 has a mesh620, the aperture diameter in the cover film is 1 mm, the aperturediameter in the PVC support is 1.6 mm, and the aperture diameter in thePSA layer is 2.6 mm and (iii) GS-SC 3 without the mesh 620, has anaperture diameter in the cover film of 1 mm, an aperture diameter in PVCsupport of 1.6 mm, and an aperture diameter of the PSA layer of 2.6 mm.

EXAMPLE 2

Example 2 discloses one embodiment for manufacturing analyte detectingdevices, more particularly electrode elements, of the present invention.It is intended to be illustrative of an embodiment, and is anon-limiting example. The batch size can be 10 sheets and include, (i)drilling apertures into P VC-support, (ii) printing conductive lines andcontrol of the resistance, (iii) printing of the insulating layer, (iv)printing reference and counter electrodes, (v) printing the workingelectrode (Composition: 50% mediator/100% buffer compounds/50% GOD),(vi) printing the hydrophilic membrane (Composition: PAA/CHAPS), (vii)printing the spacer layer In process-control with measurement ofbackground and saturation current, (viii) printing the PSA-layer, (ix)applying the mesh (for the mesh structure) and (x) applying the coverfilm 126 with two apertures. A stamping process for electrochemicalcharacterization of analyte detecting devices can include determinationof background and saturation current (n=24−48).

Measurements were taken during the maturation process, up to 3-4 weeks,directly after the maturation process and over a long period followingthe maturation process. Determination of KM as well as determination ofthe slope within the linear range 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,20, 25, 50, 100, 200, 300, 400, 500 mM glucosein buffer 20 differentglucose solutions, was done with measurement per concentration: n=8.Measurement of 40 different blood samples was performed covering aglucose range from 1 to 40 mM glucose, measurement per concentration:n=8 Evaluation. Error-Grid-Analysis was performed by applying thesamples with a pipetman and applying of the samples with finger.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols canbe made without departing from the spirit and scope of the invention

REFERENCES

The following references are incorporated herein by reference:

Adolphe X., Martemianov S., Palchetti I., Mascini M., (2005) “On theelectrochemical flow measurements using carbon-based screen-printedelectrodiffusion probes” Journal of Applied Electrochemistry 35:599-607;Jung et al. (1998), “Electrodes for electrochemical cells and method ofmaking same” U.S. Pat. No. 5,728,181;

Jarzqbek G., Borkowska Z., (1997). “On the real surface area of smoothsolid electrodes.”, Electrochimica Acta, 42(19): 2915-2918; Trasatti S.,Petri A., (1991) “Real surface measurements in electrochemistry” Pure &Appl. Chem., 63, (5): 71 -734, 1991; www.pineinst.com/echem. Accessed onMar. 5, 2008.http://www.babylon.com/definition/chronocoulometry/English. Accessed onMar. 5, 2008.

Bott A. W., Heineman W. R., (2004), “Chronocoulometry.”, CurrentSeparations, 4(20): 121-126;http://www.chem.uic.edu/chem222su05/pdf/5/StAdInt.pdf Accessed on Mar.5, 2008; and E. T. Powner, F. Yalcinkaya, (1997), “Intelligentbiosensors”, Sensor Review, 17(2):107-116.

Expected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended, therefore, that the invention be defined by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

1. A method for determining a real electrochemical surface area of aworking electrode (WE) of a screen printed sensor, comprising:determining a concentration of a mediator incorporated in a WE paste;determining a diffusion coefficient of the mediator; and determining afinal real electrochemical surface area of the WE.
 2. The method ofclaim 1, wherein the screen printed sensor is a three electrode systemthat includes, two printed carbon electrodes acting as a WE and counterelectrode, and a printed Ag/AgCl reference electrode.
 3. The method ofclaim 1, wherein the WE paste is a complex matrix structure containingboth conducting elements and assay reagents, and wherein a concentrationof the mediator incorporated in the WE paste is unknown.
 4. The methodof claim 1 wherein step (a) includes: (a) running cyclic voltammetry;and (b) applying a standard addition method.
 5. The method of claim 4,wherein in step 4(a), different solutions of mediator with differentconcentrations in PBS are prepared, cyclic voltammograms are run toobserve a (Eox)of the WE after applying increased concentrations ofmediator on the WE, and wherein a specific electrochemical responsecorresponds to an added concentration of the mediator as well as of anunknown concentration of the mediator present in the WE paste.
 6. Themethod of claim 4 wherein, step 4(b) includes, plotting oxidation peakcurrents observed in the CVs against a concentration of the addedmediator, wherein after calculating a slope and an intercept of theoxidation peak currents the unknown concentration of the mediator iscalculated by dividing the intercept with the slope.
 7. The method ofclaim 1, wherein calculation of the diffusion coefficient of themediator includes: running cyclic voltammetry under three different scanrates to create electrode cyclic voltammograms; and plotting oxidationpeak currents against a square root of scan rates.
 8. The method ofclaim 7, wherein in step 7(a) PBS is run on the electrode cyclicvoltammograms under three different scan rates to obtain three differentoxidation peak currents that correspond to the different scan rates. 9.The method of claim 7, wherein in step (7b), the slope of the plot ofthe three oxidation peak currents (Eox) against the square root of thescan rates is equal to a diffusion coefficient of the mediator.
 10. Themethod of claim 1 wherein, said step (1c) Chronocoulometry is used todetermine the real electrochemical surface area of the WE.
 11. Themethod of claim 10, wherein Chronocoulometry is a measurement of chargeas a function of time, wherein an analysis of chronocoulometric data isbased on an Anson equation that defines a charge-time dependence forlinear diffusion control:Q=2nFACD ^(1/2)π^(−1/2) t ^(−1/2) where: Q is the charge (coulombs); nis the number of electrons transferred; A is the real electrochemicalsurface area of the WE (cm²); F is Faraday's constant (96,485coulombs/mole); C is the concentration of the mediator; D is a diffusioncoefficient of the mediator (cm²/sec); and t is time (sec).
 12. Themethod of claim 11, wherein the Anson plot is a plot of Q vs. t^(1/2)and transforms data into a linear relationship whose slope (a) is equalto:2nAFCD ^(1/2)/π^(1/2) where: n is the number of electrons transferred; Ais the real electrochemical surface area of the WE (cm²); F is Faraday'sconstant (96,485 coulombs/mole); C is the concentration of the mediator(moll/cm³); and D is a diffusion coefficient of the mediator (cm²/sec).